β phosphorylation of platelet αβ is crucial for stability of arterial thrombus and microparticle formation in vivo.

BACKGROUND

It is well accepted that functional activity of platelet integrin αβ is crucial for hemostasis and thrombosis. The β subunit of the complex undergoes tyrosine phosphorylation shown to be critical for outside-in integrin signaling and platelet clot retraction ex vivo. However, the role of this important signaling event in other aspects of prothrombotic platelet function is unknown.

METHOD

Here, we assess the role of β tyrosine phosphorylation in platelet function regulation with a knock-in mouse strain, where two β cytoplasmic tyrosines are mutated to phenylalanine (DiYF). We employed platelet transfusion technique and intravital microscopy for observing the cellular events involved in specific steps of thrombus growth to investigate in detail the role of β tyrosine phosphorylation in arterial thrombosis in vivo

RESULTS

Upon injury, DiYF mice exhibited delayed arterial occlusion and unstable thrombus formation. The mean thrombus volume in DiYF mice formed on collagen was only 50% of that in WT. This effect was attributed to DiYF platelets but not to other blood cells and endothelium, which also carry these mutations. Transfusion of isolated DiYF but not WT platelets into irradiated WT mice resulted in reversal of the thrombotic phenotype and significantly prolonged blood vessel occlusion times. DiYF platelets exhibited reduced adhesion to collagen under in vitro shear conditions compared to WT platelets. Decreased platelet microparticle release after activation, both in vitro and in vivo, were observed in DiYF mice compared to WT mice.

CONCLUSION

β tyrosine phosphorylation of platelet αβ regulates both platelet pro-thrombotic activity and the formation of a stable platelet thrombus, as well as arterial microparticle release.